DNA is often methylated in normal mammalian cells. Patterns of DNA methylation have been found to correlate with patterns of gene expression. For example, differential methylation is found where maternal and paternal alleles of a gene in a zygote are differentially expressed. See Melissa Little et al., Methylation and p16: Suppressing the Suppressor, 1 NATURE MEDICINE 633 (1995). While methylation is known to occur at CpG sequences, only recent studies indicate that CpNpG sequences may be methylated. Susan J. Clark et al., CpNpG Methylation in Mammalian Cells, 10 NATURE GENETICS 20, 20 (1995). Methylation at CpG sites has been much more widely studied and is better understood.
Methylation of CpG sequences occurs by enzymatic recognition followed by placement of a methyl (CH3) group on the fifth carbon atom of a cytosine base. The enzyme that mediates methylation of CpG dinucleotides, 5-cytosine methyltransferase, is essential for embryonic development—without it embryos die soon after gastrulation. It is not yet clear whether this enzyme methylates CpNpG sites. Peter W. Laird et al., DNA Methylation and Cancer, 3 HUMAN MOLECULAR GENETICS 1487, 1488 (1994).
When a gene has many methylated cytosines it is less likely to be expressed. K. Willson, 7 TRENDS GENET. 107–109 (1991). Hence, if a maternally-inherited gene is more highly methylated than the paternally-inherited gene, the paternally-inherited gene will generally give rise to more gene product. Similarly, when a gene is expressed in a tissue-specific manner, that gene will often be unmethylated in the tissues where it is active, but will be highly methylated in the tissues where it is inactive. Incorrect methylation is thought to be the cause of some diseases, including Beckwith-Wiedemann syndrome and Prader-Willi syndrome. I. Henry et al., 351 NATURE 665, 667 (1991); R. D. Nicholls et al., 342 NATURE 281, 281–85 (1989).
The methylation patterns of DNA from tumor cells are generally different than those of normal cells. Laird et al., supra. Tumor cell DNA is generally undermethylated relative to normal cell DNA, but selected regions of the tumor cell genome may be more highly methylated than the same regions of a normal cell genome. Hence, detection of altered methylation patterns in the DNA of a tissue sample is an indication that the tissue is cancerous. For example, the gene for Insulin-Like Growth Factor 2 (IGF2) is hypomethylated in a number of cancerous tissues, such as Wilm's Tumors, rhabdomyosarcoma, lung cancer and hepatoblastomas. Rainner et al. 362 NATURE 747–49 (1993); Ogawa, et al., 362 NATURE 749–51 (1993); S. Zhan et al., 94 J. CLIN. INVEST. 445–48 (1994); P. V. Pedone et al., 3 HUM. MOL. GENET. 1117–21 (1994); H. Suzuki et al., 7 NATURE GENET 432–38 (1994); S. Rainier et al., 55 CANCER RES. 1836–38 (1995).
The present invention is directed to nucleic acids which are differentially methylated in cells exhibiting multiple drug resistance relative to drug sensitive cells. Such nucleic acids, including nucleic acids specifically disclosed herein, are isolated by virtue of differential methylation at CpG dinucleotide or CpNpG trinucleotide sequences. Surprisingly, nucleic acids disclosed herein are homologous, but not identical, to members of a known gene family, particularly the RAB6 gene. We adhere to the nomenclature whereby human genes are designated with uppercase letters (e.g., WTH3, RAB6C, RAB6), and designate encoded proteins and peptides with lowercase letters (e.g., wth3, rab6c, rab6).
Multiple drug resistances (MDR) to a broad spectrum of chemotherapeutic agents is a major obstacle in the clinical treatment of human cancer. The most extensively studied proteins, mdr1 and mrp, are structurally similar to, and are members of, the ATP-binding cassette (ABC) transporter family. Previous studies provide no cytogenetic or molecular proof of gene amplifications which might explain an elevated expression of MDR1 or MRP genes. The present invention provides nucleic acids which are homologous to the RAB6 locus. Surprisingly, the present nucleic acids were isolated because they are hypermethylated in a drug resistance cell line. Moreover, rather than being over-expressed, the present nucleic acids appear to be underexpressed in multiple drug resistance cells.